In a principal aspect, the present invention relates to a construct for protecting biologics (such as cellular materials, particularly living cellular materials) by use of three-dimensional protective structures for such biologics using generally non-reactive, three dimensional containment materials so as to enable use of such combinations in vivo and in vitro environments for growth and/or replacement of damaged or missing cellular structure such as bone.
The need for biomaterials has increased as the world population ages. Due to its chemical and crystallographic similarity to the carbonated apatite in human bones, hydroxyapatite has found applications. While sintered hydroxyapatite needs machining and is used in prefabricated forms, calcium phosphate cements can be molded as pastes and set in situ (Brown and Chow, A new calcium phosphate water setting cement, pp. 352-379 in Brown, Cements research progress, American Ceramic Society, OH, 1986; Ginebra et al., Setting reaction and hardening of an apatite calcium phosphate cement, J. Dent. Res. 76:905-912, 1997; Constantz et al., Histological, chemical, and crystallographic analysis of four calcium phosphate cements in different rabbit osseous sites, J. Biomed. Mater. Res. [Appl. Biomater]. 43:451-461, 1998; Miyamoto et al., Histological and compositional evaluations of three types of calcium phosphate cements when implanted in subcutaneous tissue immediately after mixing, J. Biomed. Mater. Res. [Appl. Biomater.] 48:36-42, 1999; Lee et al., Alpha-BSM(R): A biomimetic bone substitute and drug delivery vehicle, Clin. Orthop Rel. Res. 367:396-405, 1999). Calcium phosphate cement, referred to as CPC, is comprised. of a mixture of fine particles of tetracalcium phosphate [TTCP: Ca4(PO4)2O] and dicalcium phosphate anhydrous [DCPA: CaHPO4]. The CPC powder can be mixed with water to form a paste that can intimately conform to osseous defects with complex shapes and set in vivo to form a hydroxyapatite-containing implant. Due to its in-situ setting ability, excellent osteoconductivity and bone replacement capability, CPC is promising for a wide range of clinical applications.
The term “in-situ setting” or “self-hardening” or “moldable” refers to the paste being able to set or harden inside a bone cavity or in a mold and being able to be shaped or contoured; for example, the CPC paste can be placed into a bone cavity and harden when in contact with an aqueous medium (Matsuya et al., Effects of mixing ratio and pH on the reaction between Ca4[PO4]2O and CaHPO4, J. Mater. Sci.: Mater. in Med. 11:305-311, 2000; Takagi et al., Morphological and phase characterizations of retrieved calcium phosphate cement implants, J. Biomed. Mater. Res. [Appl. Biomater.] 58:36-41, 2001). U.S. Pat. Nos. 5,525,148, 5,545,254, 5,976,234, and 5,997,624 (Chow et al.) disclose methods for calcium phosphate cements including fillers and pore forming agents. Xu et al. suggest fiber reinforcement of calcium phosphate cement in “Reinforcement of a self-setting calcium phosphate cement with different fibers”, J. Biomed. Mater. Res. 52:107-114 (2000) and in “Effects of fiber length and volume fraction on the reinforcement of calcium phosphate cement”, J. Mater. Sci.: Mater. in Med. 12:57-65 (2001). Von Gonten et al. suggest a single sheet of mesh reinforcement for calcium phosphate cement in “Load-bearing behavior of a simulated craniofacial structure fabricated from a hydroxyapatite cement and bioresorbable fiber-mesh”, J. Mater. Sci.: Mater. in Med. 11:95-100 (2000). However, in these studies, there has been no mention of incorporating living cells into CPC. Takagi et al. suggest the formation of macropores resulting from the dissolution of soluble fillers or pore forming agents in “Formation of macropores in calcium phosphate cement implants”, J. Mater. Sci. Mater. in Med. 12:135-139 (2001). Chow reviews calcium phosphate cements in “Calcium phosphate cements: Chemistry, properties, and applications”, Mat. Res. Symp. Proc. 599:27-37 (2000). Xu et al. incorporate fibers and pore forming agents in “Strong and macroporous calcium phosphate cement: Effects of porosity and fiber reinforcement on mechanical properties”, J. Biomed. Mater. Res. 57:457466 (2001). Xu et al. use resorbable fibers in “Calcium phosphate cement containing resorbable fibers for short-term reinforcement and macroporosity”, Biomaterials 23:193-202 (2002). Xu et al. use synergistic reinforcement to develop scaffolds in “Effects of Synergistic reinforcement on in situ hardening calcium phosphate composite scaffold for bone tissue engineering” Biomaterials 25:1029-1037 (2004). Xu et al. also develop fast-setting and anti-washout CPC in “Fast-setting and anti-washout calcium phosphate scaffolds with high strength and controlled macropore formation rates”, Journal of Biomedical Materials Research 68A:725-734 (2004). However, in these studies there is no mention of mixing living cells into the CPC paste.
Cell culture studies were performed on CPC to investigate the biocompatibility of CPC (Simon et al., Preliminary report on the biocompatibility of a moldable, resorbable, composite bone graft consisting of calcium phosphate cement and poly[lactide-co-glycolide] microspheres. J Orthop Res 20:473-482, 2002). Cell culture was performed to investigate the biocompatibility of CPC-fiber composite by using osteoblast-like cells (Xu et al., Self-hardening calcium phosphate composite scaffold for bone tissue engineering. Journal of Orthopaedic Research 22:535-543, 2004), CPC-mesh composite (Xu et al., Self-hardening calcium phosphate cement-mesh composite: Reinforcement, macropores, and cell response. Journal of Biomedical Materials Research 69A:267-278, 2004), and fast-setting CPC composite (Xu et al., Fast-setting calcium phosphate-chitosan scaffold: Mechanical properties and biocompatibility, Biomaterials, in review, 2004). Cells were seeded onto the specimens surfaces. The cell attachment, live and dead cell staining, and cell viability were investigated for these compositions. However, in these studies, no cells were mixed into the CPC paste. The CPC specimens were set or hardened without any cells in them. The cells were subsequently attached to the surfaces of the already hardened specimens.
U.S. Pat. No. 4,353,888 (Sefton) discloses encapsulation of viable mammalian cells to form beads for introduction into a host body. U.S. Pat. No. 4,892,538 (Aebischer et al.) discloses in vivo delivery of neurotransmitters by implanted, encapsulated cells using a semipermeable membrane which permits the diffusion of the neurotransmitter while excluding viruses and antibodies. U.S. Pat. No. 6,132,463 (Lee et al.), U.S. Pat. No. 6,139,578 (Lee et al.), U.S. Pat. No. 6,277,151 (Lee et al.), and U.S. Pat. No. 6,544,290 (Lee et al.) disclose a poorly-crystalline apatitic calcium phosphate material seeded with cells for bone and cartilage growth, in vitro cell culture systems and cell encapsulation matrices. U.S. Pat. No. 6,143,293 (Weiss et al.) discloses in-direct pre-formed scaffolds that can be seeded with cells: U.S. Pat. Application 2004/0101960 A 1 (Schaefer et al.) discloses an injectable hydroxyapatite cement containing living cells as a bone substitute material. Simon et al. (Cell seeding into calcium phosphate cement, Journal of Biomedical Materials Research, 68; A:628-639, 2004) show cell encapsulation in alginate beads that are then mixed with a CPC paste. None of this prior art mentions the design of three-dimensional cell protectors of lattice-like structures, or zigzag or woven or curved structures, or three-dimensional web-like structures. Furthermore, none of this prior art mentions the development of cell protectors for two functions: (1) protecting the cells from the surrounding environment for a predetermined time period; (2) controlled formation of three-dimensional pore architectures when the cell protectors dissolve and concomitantly release the living cells into the highly-interconnected pore structures in the in-situ setting implant.
U.S. Pat. Application 2002/0055143 A1 (Bell et al.) discloses bone precursor compositions including an injectable calcium cement than can be conditioned with cells, preferably bone tissue cells. It does not mention the use of three-dimensional cell protectors/pore architecture builders. U.S. Pat. Application 2003/0019396 A 1 (Edwards et al.) discloses a porous cement which self sets to hydroxyapatite and has an interconnected porosity. It does not mention the mixing of cells into the cement paste. U.S. Pat. Application A1 2003/0099630 (DiBenedetto et al.) discloses a bioactive material using fibroin solutions and suspensions that is injectable and able to form pores, and can release compounds in a controlled manner to enhance growth and activation of cells. It does not mention the mixing of cells into the injectable composition and the use of cell protectors/pore architecture builders.
U.S. Pat. Application 2003/0206937 A 1 (Gertzman et al.) discloses a malleable bone putty including a bone powder mixed in a hydrogel carrier. U.S. Pat. Application 2004/0062809 A1 (Honiger et al.) discloses porous biocompatible polymers in the form of hydrogels with a three-dimensional structure and communicating cells. Its three-dimensional structure refers to the structure of the porous implant as a whole; it does not refer to the formation of the pores alone. It does not mention the use of cell protectors/pore architecture builders incorporated inside an in situ implant matrix material.
U.S. Pat. Application 2004/0101518 A1 (Vacanti et al.) discloses a hydrogel-cell composition with a support structure having a predetermined shape that corresponds to the shape of a desired tissue. It does not use the hydrogel to protect the cells and form pore structures in the final implant. Neither does it mention the use of cell protectors and pore architecture builders that are incorporated inside an in situ implant matrix material.
U.S. Pat. Application 2004/0131678 A1 (Burger et al.) discloses a water based bone cement comprising a slow release bone growth factor and a fast release antimicrobial agent. U.S. Pat. Application 2004/0137032 A1 (Wang) discloses combinations of calcium phosphates, bone growth factors, and pore-forming additives as osteoconductive and osteoinductive composite bone grafts. It relates to the degradation of polymer microspheres as porogens leading to macropores that facilitate the growth of osteoblasts into the bone grafts. The cells are grown into the pores of the graft after the paste setting; the cells are not mixed into the paste prior to the paste setting. In this application the cells attach to a set and hardened surface. Furthermore, the polymer microspheres and/or water-soluble particles create pores that are sphere-like, discrete, or particle-shaped. It does not mention the use of cell protectors of three-dimensional lattice-like structures, or zigzag or woven or curved structures, or three-dimensional web-like structures. Nor does it mention the three-dimensional pore architecture builders where the cells are incorporated throughout the entire interior of the implant prior to the paste setting.
In conclusion, in prior art, there has been no mention of methods of fabricating surgical moldable and self-hardening implants containing living cells prior to paste placement where said living cells are protected in three-dimensional cell protectors for a prescribed time period, said protectors then subsequently dissolve at a predetermined time to free the cells inside the implant and concomitantly form three-dimensional pore architectures as cell and nutrient passageways throughout the entire interior of the implant.
Furthermore, there has been no mention in prior art of the design of cell protectors/pore architecture builders of three-dimensional lattice-like structures, zigzag or woven or curved structures, or three-dimensional web-like structures inside surgically placed implants. These structures serve two important functions: (1) protecting the cells, for a controlled period of time, from any undesirable environment including mechanical damage, pH changes, ion activities, and excessive temperature changes during paste setting; and (2) at a prescribed time, creating engineered three-dimensional pore architectures as cell nutrient and communication passageways throughout the implant.
In addition, in prior art, there has been no mention of using three-dimensional cell protectors/pore architecture builders inside a surgical moldable and self-hardening implant, with three-dimensional cell protectors/pore architecture builders having designed sizes, effective diameters, interconnectivity sizes, and degradation rates matching the applications, so that the cells can be released and pores created at a controlled time or at graded times.